Isotopologue fractionation during N2O production by fungal denitrification

Identifying the importance of fungi to nitrous oxide (N₂O) production requires a non‐intrusive method for differentiating between fungal and bacterial N₂O production such as natural abundance stable isotopes. We compare the isotopologue composition of N₂O produced during nitrite reduction by the fungal denitrifiers Fusarium oxysporum and Cylindrocarpon tonkinense with published data for N₂O production during bacterial nitrification and denitrification. The fractionation factors for bulk nitrogen isotope values for fungal denitrification were in the range −74.7 to −6.6‰. There was an inverse relationship between the absolute value of the fractionation factors and the reaction rate constant. We interpret this in terms of variation in the relative importance of the rate constants for diffusion and enzymatic reduction in controlling the net isotope effect for N₂O production during fungal denitrification. Over the course of nitrite reduction, the δ¹⁸O values for N₂O remained constant and did not exhibit a relationship with the concentration characteristic of an isotope effect. This probably reflects isotopic exchange with water. Similar to the δ¹⁸O data, the site preference (SP; the difference in δ¹⁵N between the central and outer N atoms in N₂O) was unrelated to concentration during nitrite reduction and, therefore, has the potential to act as a conservative tracer of production from fungal denitrification. The SP values of N₂O produced by F. oxysporum and C. tonkinense were 37.1 ± 2.5‰ and 36.9 ± 2.8‰, respectively. These SP values are similar to those obtained in pure culture studies of bacterial nitrification but quite distinct from SP values for bacterial denitrification. The large magnitude of the bulk nitrogen isotope fractionation and the δ¹⁸O values associated with fungal denitrification are distinct from bacterial production pathways; thus multiple isotopologue data holds much promise for resolving bacterial and fungal production. Our work further provides insight into the role that fungal and bacterial nitric oxide reductases have in determining site preference during N₂O production. Copyright © 2008 John Wiley & Sons, Ltd.